Synthetic small duplex RNAs complementary to gene promoters within chromosomal DNA are potent inhibitors or activators of target gene expression in mammalian cells (Morris et al., 2004; Ting et al., 2005; Janowski et al., 2005; Li et al., 2006; Janowski et al., 2007). These synthetic RNAs are called antigene RNAs (agRNAs) to distinguish them from small duplex RNAs that target mRNA. agRNAs recruit members of the argonaute (AGO) protein family to RNA transcripts that originate from the target gene promoter (Janowski et al., 2006; Kim et al., 2006; Han et al., 2007; Schwartz et al., 2008). Recognition of the target RNA occurs in close proximity to the chromosome, resulting in transcriptional modulation of the target gene.
One remarkable feature of the synthetic agRNAs that the inventors have examined is the potency and robustness of their activity when they are introduced into cells. This potency, coupled with the presence of protein machinery that facilitates their function, suggests that endogenous small RNAs may possess the ability to recognize gene promoters. If RNA could direct proteins to specific gene promoters, such RNA-mediated modulation of transcription might have evolutionary advantages relative to the development of gene-specific protein transcription factors.
Synthetic duplex RNAs that are complementary to mRNA (small interfering RNAs or siRNAs) are also potent and robust agents for modulating gene expression (Fire et al., 1998). siRNAs are known to have endogenous analogs that regulate gene expression called microRNAs (miRNAs) (Lagos-Quintana et al., 2001). miRNAs are processed inside the cell from RNA precursors that contain stem-loop structures. These stem-loop structures are processed by the double-stranded nucleases Drosha and Dicer to produce mature miRNAs.
As of the current release of the miRNA repository (miRBase v12.0), 866 human miRNAs have been annotated, but this number continues to increase. Several miRNAs that recognize sequences within the 3′-untranslated regions (3′UTR) of mRNA transcripts have been characterized. Many miRNAs, however, have no known targets (Lee et al., 1993; John et al., 2004) while some can recognize multiple mRNAs, suggesting that the determinants of miRNA interactions are complex and poorly understood.
Two reports based on computational analyses have suggested that miRNAs can modulate gene expression through promoter recognition. Dahiya and co-workers used publicly available software (RegRNA) to search for potential miRNA target sites within the promoter of the E-cadherin gene (Place et al., 2008). They identified one potential binding site for miR-373 within the E-cadherin promoter and reported that introduction of a synthetic miR-373 mimic increased expression of the gene by 6-fold at the level of the mRNA. Rossi and co-workers searched for perfect complementarity between miRNAs and gene promoters (Kim et al., 2008). Their analysis suggested that miR-320 targets the genomic location from which it is transcribed and showed that expression of miR-320 and the adjacent gene, POLR3D, are anti-correlated.
The above-mentioned studies either analyzed a single gene promoter or used highly stringent sequence comparison criteria. These approaches were not intended to assess the broader potential for miRNAs to recognize gene promoters, warranting a more thorough evaluation of the relationship between miRNAs and promoter sequences.
A practical justification for more comprehensive studies is that validating natural gene targets of miRNAs is a complex and difficult process. The development of systematic and efficient methods for identifying promoter sequences that may be miRNA targets is essential for prioritizing predictions and efficiently allocating experimental resources towards validating the most promising targets.